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Title: Characterisation and structure/function studies of the liver-type and brain-type glucose transporters
Author: Seatter, Michael John
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1994
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Km values were measured for zero-trans entry of 2-deoxy-D-glucose and beta-D-fructose into Xenopus oocytes heterologously expressing GLUT2. GLUT2 transports 2-deoxy-D-glucose with a Km of 11 mM and D-fructose with a Km of 67 mM. Ki values for D-glucose, maltose, D-fructose and cytochalasin B inhibition of 2-deoxy-D-glucose transport by GLUT2 were measured. Inhibition of 2-deoxy-D-glucose transport by D-glucose, maltose and D-fructose was competitive, with Ki values of 6.5 mM, 125 mM and 200 mM, respectively. Inhibition by cytochalasin B was non-competitive, with a Ki of 6.9 muM. In addition, Ki values were measured for D-glucose, maltose, and cytochalasin B inhibition of D-fructose transport by GLUT2. D-glucose and maltose were competitive inhibitors displaying Ki values of 15.3 mM and 116 mM, respectively. Inhibition by cytochalasin B was non-competitive, with a Ki value of 6.1 muM. The inhibitory effects of L-sorbose and 2,5-anhydro-D-mannitol, beta-D-fructopyranose and beta-D-fructofuranose analogues, respectively, on 2-deoxy-D-glucose transport into Xenopus oocytes by heterologously expressed GLUT2, were investigated. L-sorbose and 2,5-anhydro-D-mannitol both competitively inhibited 2-deoxy-D-glucose transport with Ki values of 170 mM and 26 mM, respectively, suggesting that beta-D-fructose is likely to interact with GLUT2 in the furanose ring form. The structural basis of sugar binding to GLUT2 has been investigated using a range of hexoses, halogeno- and deoxy-D-glucose analogues to inhibit the transport of 2-deoxy-D-glucose by GLUT2 expressed heterologously in Xenopus oocytes. Results indicate that hydrogen bonding is important at the C-1, C-3, and to a lesser extent at the C-4 and C-6 hydroxyls of D-glucose, and that hydrophobic interactions at the C-6 position may also prove important. The results obtained for GLUT2 have been compared with those published for GLUT1 and GLUT4, and with GLUT3 in oocytes, and the binding patterns of the transporters were found to be broadly similar but this varied subtley between isoforms. A model has been produced to show the interactions of D- glucose and D-fructose with the exofacial binding site of GLUT2. Site-directed mutagenesis and recombinant polymerase chain reaction (PCR) technology have been used to construct a series of nineteen mutant GLUT3 cDNAs. These contain single codon changes such that each cDNA encodes a protein with a single transmembrane helix 8 residue substituted with alanine. These transporter cDNAs have been used as a template for in vitro synthesis of mRNA which has been injected into Xenopus oocytes. Each of these mutants has been found to transport 2-deoxy-D-glucose at levels between 40-200% of that measured with wild-type GLUT3, but expression levels have not yet been determined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available